Our Abpromise guarantee covers the use of ab3583 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ICC/IF Use a concentration of 5 µg/ml.
Inhibition Assay Use at an assay dependent concentration.
ICC 1/50 - 1/200.
IHC-Fr 1/50 - 1/200.
WB 1/200 - 1/1000. Detects a band of approximately 16 kDa (predicted molecular weight: 16 kDa).
Flow Cyt Use 3-5µl for 106 cells.



  • ab3583 at a dilution of 1 / 4000 staining leptin in mouse 3T3-L1 cell lysate by Western blot.

  • ab3583 staining Leptin in 3T3-L1 cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 1% BSA for 1 hour at room temperature. Samples were incubated with primary antibody (2ug/ml in 0.1% BSA) for 3 hours at room temperature. An Alexa Fluor® 488-conjugated Goat anti-rabbit IgG (H+L) polyclonal was used as the secondary antibody (1/2000) (Panel a). Nuclei were stained with DAPI (Panel b). F-actin stained with Rhodamine Phalloidin (panel c). Merged images shown in panel d. 

  • ICC/IF image of ab3583 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab3583, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • ab3583 staining Leptin in Human mesenchyml stem cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and blocked with 10% serum for 30 minutes at 22°C. Samples were incubated with primary antibody (1/100 in 10% horse serum) for 1 hour at 22°C. A FITC-conjugated Mouse anti-rabbit IgG1 monoclonal was used as the secondary antibody (1/100).

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  • Immunohistochemical staining of Leptin in Sheep brain using ab3583.

  • ab3583 staining Leptin in 3T3-L1 cells by Flow Cytometry. The sample was incubated with the primary antibody (3ug/ml in 2.5% BSA) for 2 hours at room temperature. An Alexa Fluor 488®-conjugated Goat anti-rabbit (1/400) was used as the secondary antibody. Red histogram represents ab3583, pink histogram represents isotype control, purple histogram represents unstained control, green histogram represents no primary antibody control


This product has been referenced in:
  • Han YC  et al. Leptin regulates disc cartilage endplate degeneration and ossification through activation of the MAPK-ERK signalling pathway in vivo and in vitro. J Cell Mol Med N/A:N/A (2018). WB . Read more (PubMed: 29372627) »
  • Liu Y  et al. HN1L Promotes Triple-Negative Breast Cancer Stem Cells through LEPR-STAT3 Pathway. Stem Cell Reports 10:212-227 (2018). Read more (PubMed: 29249663) »

See all 15 Publications for this product

Customer reviews and Q&As

Abcam has not validated the combination of species/application used in this Abreview.
Western blot
Spermophilus tridecemlineatus Tissue lysate - whole (white adipose tissue)
Gel Running Conditions
Reduced Non-Denaturing (Native) (12.5)
Loading amount
20 µg
white adipose tissue
Blocking step
BSA as blocking agent for 15 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Feb 05 2014

Thank you for contacting us. This antibody has not been tested for blocking functional activity. If you do choose to test it this we way encourage you to submit a review to let us and our customers know whether it worked for you in functional studies....

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Abcam guarantees this product to work in the species/application used in this Abreview.
Immunocytochemistry/ Immunofluorescence
Human Cell (Mesenchymal stem cell)
Mesenchymal stem cell
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 22°C

Mr. Steven Johnstone

Verified customer

Submitted Sep 05 2012

Thank you for your enquiry.

I can confirm that ab3583 and ab16227 antibodiesshould detect leptin both intra and extracellularlly.However based on the references for these product it appears that most if not all of the leptin is immediately s...

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Thank you for passing some information to me.
I understand that ab3583 has been used previously at 1:4000.
Have you had a chance to test this antibody at dilutions of 1/500 or 1/1000 (for 1 hr at room temperature or overnight at 4oC?

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Thank you for getting back to me and for specifying the sample types.
As you have kindly explained to me in your previous e-mail, the primary and the secondary antibodies worked with the positive control but the signal was very weak using lysates ...

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Thank you for confirming the order dfetails.
Would you be so kind to clarify what samples you have been using:
- species,
- cell or tissue types,
- lysate (total cell lysates, or cytoplasmic fraction etc)
I look forward to hear...

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Thank you for your prompt answers. Your co-operation in this matter is highly appreciated.
I understand that you have already carried out some optimization steps and it seems that the primary and the secondary antibodies worked on positive control...

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Thank you for enquiry and for taking the time to provide some useful details of the experiments. As I understand three of our products (ab3583, ab13987 and ab97051) have been used in these studies.
Though you have kindly provided some details, it ...

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Thank you for contacting us.

I am sorry that the vial you received did not contain the full amount of the product and I apologize for the inconvenience.We will be pleased to issue a free of charge replacement vial. In order to do this, I am ...

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